Phosphorylation Of Liver Glycogen Synthase Research Articles

In vivo phosphorylation of liver glycogen synthase. Effect of glucose and glucagon treatment of liver cells on the distribution of the [32P]phosphate.

Glycogen synthase was phosphorylated in vivo by perfusing rat liver or incubating liver cells with [32P]phosphate. It was then isolated by immunoprecipitation and subjected to exhaustive tryptic proteolysis. The trypsin-derived [32P]phosphopeptides were separated by high pressure liquid chromatography (HPLC). Incubation of in vivo phosphorylated synthase with endogenous synthase phosphatase to convert synthase D to synthase R resulted in removal of phosphate from all of the labeled phosphopeptides. In prelabeled liver cells treated with glucagon or glucose, the activities of synthase and phosphorylase changed in the direction expected. The total labeling in the immunoprecipitated synthase was found to be increased to 126% and decreased to 67% of the control with glucagon and glucose treatment, respectively. When the HPLC [32P]phosphopeptide profile of synthase from glucagon-treated animals was compared with that of controls, there were only minor differences in the two profiles. All the peaks were present and the proportion of labeling in each remained similar. There also was only a modest change in the [32P]phosphopeptide profile with glucose treatment when compared with that of controls. These results indicate that regulation of synthase activity in the hepatocyte involves changes in phosphorylation at multiple sites. Indeed, in 32P-labeled liver cells, all of the labeled sites appeared to be involved.

Role of protein kinase C in the regulation of rat liver glycogen synthase

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca 2+- and phospholipid-dependent fashion to 1–1.4 mol PO 4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca 2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.

Threonine phosphorylation of rat liver glycogen synthase

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [ 32P] phosphoserine, significant levels of [ 32P] phosphothreonine (7% of the total [ 32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [ 32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 “in vitro” (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the “in vivo” phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.

In vivo phosphorylation of liver glycogen synthase. Isolation of the 32P-labeled enzyme and studies on the nature of the bound [32P]phosphates.

Our previous study (Tan, A. W. H., and Nuttall, F. Q. (1983) J. Biol. Chem. 258, 9624-9630) indicated that liver synthase D contained a large number of endogenous phosphates, 12 of which were stable and 6 labile to alkali treatment. We wished to investigate the nature of the phosphates on synthase which became isotopically labeled when inorganic [32P]phosphate was given either to intact rats or to isolated liver cells. An antibody against liver synthase D was used for the isolation of synthase. The antibody recognized both the phosphorylated and dephosphorylated form of the enzyme, native as well as partially cleaved species. A large enzyme form, with Mr of 90,000 as well as one with Mr of 73,000 was observed. A 61% decrease in [32P]phosphate was found in synthase when prelabeled liver cells were treated with glucose, whereas a 25% increase was seen in cells treated with glucagon. After [32P]synthase D was converted to synthase I by synthase phosphatase, 95% of the [32P]phosphate was lost. All of the bound [32P]phosphates were found to be labile to alkali. Thus, under the in vivo conditions used, the [32P]phosphates incorporated into synthase were characterized by their fast turnover rate, alkali lability and susceptibility to the action of synthase phosphatase, both in vivo and in vitro. These criteria serve to distinguish them from the slower turning-over, alkali-stable phosphates found previously in both synthases D and I.

Phosphorylation and inactivation of rat liver glycogen synthase by cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1.

Phosphorylation of liver glycogen synthase by cAMP-dependent protein kinase (A-kinase) results in the incorporation of approximately 0.7 to 1.0 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and peptide mapping reveal the presence of a single 32P-labeled peptide. This extent of phosphorylation does not result in a significant reduction of the synthase activity ratio. Phosphorylation of the liver synthase by cAMP-independent synthase (casein) kinase-1 results in the incorporation of 1.6 to 2.2 mol of PO4/subunit. Although at least 4 tryptic peptides have been found to be labeled with 32P, no significant reduction of the synthase activity ratio was observed. Under the same assay conditions, the muscle synthase is effectively inactivated by either kinase alone or in combination. Inactivation of liver synthase can be achieved after phosphorylation by A-kinase and followed by synthase (casein) kinase-1. However, the inactivation becomes less effective if the order of the addition of these two kinases is reversed. Under the latter assay condition, the phosphate incorporation is less than additive in the presence of both kinases. Prior phosphorylation of the synthase by A-kinase transforms the synthase to become a better substrate for synthase (casein) kinase-1 as evidenced by a 3- to 5-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase by synthase (casein) kinase-1 results from the rapid phosphorylation of a site neighboring to that previously phosphorylated by A-kinase.